Androgens constitute a group of 19-carbon steroid hormones that exert profound influence on the male genital tract and are involved with the development and maintenance of secondary male sex characteristics such as beard growth, deepening of the voice at puberty, muscle and bone development, body strength, and sexual drive. Androgens are synthesized in the male testis, in the female ovary, and in the adrenal cortex of both sexes. Once released into the blood circulation, these endogenous androgens serve both as hormones and as prohormones for the formation of two different classes of steroids: 5.alpha.-reduced androgens, which act as the intracellular mediators of most actions of the male sex hormones; and estrogens, which enhance some androgenic effects and block others.
Testosterone is the principal androgen secreted by the testis and is the main androgenic steroid in the plasma of males. In women, small amounts of testosterone are synthesized by the ovary and adrenals. Typically, testosterone is reduced at the 5.alpha. position into dihydrotestosterone, which serves as the intracellular mediator of most hormone actions. Although a variety of other naturally occurring androgens have been identified, these are generally weak in potency; and it is now generally believed that they are androgens only to the extent that they can be converted in-vivo to testosterone and/or dihydrotestosterone.
The major metabolites of androgens in urine are physiologically inactive either as free steroids or as water-soluble conjugates. These metabolites are predominantly etiocholanolone, a 5.alpha.-reduced metabolite of testosterone; and androsterone, a metabolite of dihydrotestosterone. It is now also recognized that testosterone (but not dihydrotestosterone can be aromatized into estradiol in a variety of extraglandular tissues, a pathway that accounts for most estrogen synthesis in men and postmenopausal women. The role, if any, of the approximately 50 micrograms of estradiol synthesized each day in normal men has never been defined. Nevertheless, the production of estradiol is considered a normal phenomenon. Experimental evidence suggests that estradiol affects the proliferation of male sex secondary organs; and that estradiol is necessary to induce prostate cancer in animal models. For a more detailed and comprehensive description of in-vivo synthesis of androgens, its metabolic pathways, intermediates, and reaction products, the following texts are recommended: The Pharmacological Basis Of Therapeutics (Goodman et al., editors), 7th edition, MacMillan Publishing Company, New York, 1985, pages 1440-1458; Basic & Clinical Endocrinology (Greenspan and Forsham, editors), 2nd edition, Lange Medical Publications, Los Altos, California, 1986.
The elucidation of the chemistry of androgens found in-vivo and the evaluation of androgenic potency for such compositions were and remain based on the use of specific in-vivo bioassays. The classical bioassay is based upon the growth of the comb of the capon, an assay developed by A. Butenandt and Tscherning, K. 8 Z. Physiol. 229:167 (1934)]. The determination of androgenic activity utilizing the growth response of the capon's comb was employed in the first isolation of androsterone, a metabolite of testosterone, from male urine [Butenandt, A., Z. Angew. Chem. 4:905-908 (1931)]. Subsequently, bioassays using live mammals in which the test depended upon inducing the growth of the seminal vesicles or ventral prostate of the castrated male were found to provide better correlations of activity and potency. These in-vivo mammalian bioassays were employed for the evaluation of testicular extracts [Loewe, S. and H.E. Voss, Klin. Wochenschr. 9:481-487 (1930)]; and for the isolation in crystalline form and elucidation of chemical structure for testosterone [David et al., Hoppe Seylers Z. Physiol. Chem. 233:281-282 (1935); Ruzicka, L. and A. Wettstein, Helv. Chim. Acta 18:1264-1275 (1935)]. Moreover, these in-vivo bioassays remain the most reliable methods known today by which to evaluate the androgenic potency of different chemical compositions.
In actual practice, these in-vivo animal tests measure the increase in wet weight of the ventral prostate or the seminal vesicles of castrated animals based upon the administration of androgenic hormones. It is now known that androgens induce cell proliferation, that is--an increase in the cell number (hyperplasia); an cause an enlargement of individual cell size (hypertrophy); and also cause the accumulation of water (water imbibition) and solutes in the extracellular space.
Accordingly, it is recognized and appreciated that the total wet weight does not represent an appropriate estimate of only the proliferative action of hormone at all. In addition to these hormonal actions, androgens also subsequently inhibit the proliferation of their target cells in-vivo; it is noted that once the prostate and seminal vesicles of the castrated animal reach the cell number found in normal adult males during continued administration of testosterone, a plateau level in cell number is reached and no further cell proliferation is observed [N. Bruchovsky et al., Vit & Horm. 33:61-102 (1975)]. In sum, therefore, these in-vivo animal assays measure only the entire combination of all these diverse hormonal effects and cannot effectively measure only the proliferative capability alone.
In an effort to avoid using live animals and to diminish the variability of in-vivo assay methods, a variety of in-vitro tests have been proposed. These include: The assay of sex hormone-binding globulins; and specific radioimmunoassays sensitive for androgens [James et al., "Measurement Of Androgens In Biological Fluids", in Androgens And Antiandrogens (Maritini, L. and M. Motta, editors), Raven Press, New York, 1977, pages 19-35]. Similarly, the search for non-androgenic anabolic steroids has made use of in-vitro assays employing the growth of the kidney or levator ani muscle of castrated animals; and introduced in-vitro assays examining nitrogen excretion and the nitrogen-retaining effects of agents given to live animals on controlled diets. As recognized generally, none of these substitute in-vitro assays have been satisfactory.
It is also noteworthy that all the presently accepted assay techniques, whether performed in-vivo or in-vitro, are based upon a single common mechanism of action for all androgens as a class--including endogenous androgens; synthetically synthesized androgens; and other substances demonstrating the requisite ability to develop and maintain secondary male sex characteristics which is the classical definition and identifying trait of an androgen. The theory almost universally accepted today relies upon the assumption that androgenic effects can only be mediated by the interaction of androgens with intracellular androgen receptors present in the testis and accessory structures (epididymis, vas deferens, and seminal vesicles); the sebaceous glands; and the skeletal muscles. Potent androgens, such as testosterone or dihydrotestosterone, are said to bind to such a cytoplasmic protein receptor and to form a hormone-receptor complex intracellularly. This complex once formed then is said to be physically translocated into the nucleus of the cell where further binding of the complex at specific sites on the chromosomes is said to occur. This in turn is said to cause a general increase in protein synthesis and is said also to initiate new proliferation of the cells in-situ. Accordingly, the conventional mechanism of action for androgens as a class is that all the properties and known actions for androgens are mediated by a single class of cytoplasmic receptor protein; and that it is impossible to separate between the properties of new cell proliferation and new protein synthesis provided by an androgen because the mechanism of action is the same regardless of intended biological or clinical effects [Mainwaring, W.I.P., Monogr. Endocrinol. 10:1-178 (1977); Pardridge, W.M., Endrocrin. Rev. 103-123 (1981); The Testis (Burger, H. and D. DeKretser, editors), Raven Press, New York, 1981; Moguilewsky et al., in Prostate Cancer, Part A, Alan R. Liss, Inc., 1987, pp. 315-340].
With this conventional mechanism of action in mind, the recognized therapeutic uses for endogenous and synthetic androgens lie primarily in treating deficient endocrine function of the testis. In addition, some potent androgens have been utilized in treating a variety of other clinical disorders in the hope that their administration on non-genital tissues would also be beneficial. These other clinical conditions include: hypogonadism; improvements in nitrogen balance and muscle development, particularly via the use of anabolic steroids; the stimulation of erythropoiesis; hereditary angioneurotic edema; the management of growth retardation and short stature; carcinoma of the female breast; osteoporosis; and decreasing total plasma triglycerides and very low density lipoproteins in the blood.
Complementary to the use of known potent androgens has been the search for compounds which might actually inhibit or neutralize the action of androgens--the so-called "antiandrogens". The treatment of cancer of the prostate was clearly one of the earlier aims and goals for using such antiandrogens; however, the therapeutic uses of potent antagonists of androgens have been suggested for virilization in women, precocious puberty in boys, acne treatment, and satyriasis in adult men. There is also considerable interest in the potential use of such antiandrogens as male contraceptives. Substative progress along these lines, however, has been much less than might be hoped for.
Antiandrogens have been classified as steroidal (exemplified by cyproterone acetate and medroxyprogesterone acetate) and as non-steroidal [exemplified by flutamide and its metabolite, hydroxy-flutamide, and anandron (R23908); Raynaud and Ojasoo, J. Ster. Biochem. 25:811-833 (1986)]. Probably the best known and frequently employed potent steroidal antiandrogen is cyproterone acetate [Neri, R.O., Adv. Sex Horm. Res. 2:233-262 (1976);; Neumann, F., Ir. J. Med. Sci. 15:61-70 (1982)]. A fairly comprehensive listing of presently known antiandrogens can be found in Prostate Cancer, Part A, (Murphy, G.P. et al., editors), Alan R. Liss, New York, 1987; and Raynaud, J.P. and Ojasoo, T., J. Ster. Biochem. 25:811-833 (1986). The difficulty with these conventionally known compositions is that when these putative antiandrogens are given therapeutically, they may affect a variety of different endocrine organs such as the hypophysis, the adrenal cortex, and the testis indiscriminately and through these actions, these compositions may decrease plasma androgen levels. Note that true antiandrogens should impair only the effects of potent androgens rather than cause an actual decrease in androgen production and a reduction of plasma androgens. Unfortunately, the presently known antiandrogens appear not to discriminate between these vastly different properties and capabilities; and, accordingly, remain highly questionable in their abilities as true antiandrogens.
The difficulties of identifying and evaluating new compositions which are agonists and antagonists of androgens are generally recognized despite the variety and disparity of methods known to date. In-vivo methods have been generally difficult to perform in a consistent manner; and are open to independent biological variations which cannot be entirely controlled or eliminated. Conventionally available in-vitro systems have been at best indirect assessments or estimates of many different properties and parameters which are at best only somewhat correlatable with the potency and in-vivo activity of known androgens as a class. Equally limiting and restrictive is the continuing demand and reliance upon a generally acceptable single mechanism of action for androgenic compounds and antiandrogenic compositions--the pharmacological and biological effects for which must be tailored to conform and comply with an often contradictory and extremely complex series of cellular events inherently based on the existence and use of cytoplasmic receptor protein specific for these compositions. As can now be readily appreciated, there is a long standing and continuing need for reliable, accurate, and sensitive in-vitro assay protocols which will identify those pharmacologically active substances which are demonstratably potent as androgens and which will distinguish these from inhibitors or neutralizers of potent androgens, and from those substances which are completely quiescent with respect to the proliferative effect of androgens.